The existence of tumor-initiating cancer stem cells (CSC) has been shown in a variety of solid tumors (e.g. breast, prostate, glioblastoma, liver). However, these CSC have highly variable antigenic and functional properties even when derived from the same tumor. These observations highlight a cardinal problem in CSC biology, namely, the heterogeneity of the CSC. Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all three mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSCs and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Although a variety of different stem cell markers have been used to isolate and characterize CSC from HCC, none of them seemed to be specific for liver malignancies and none of the isolated fractions showed uniform properties. Therefore we opted to use a functional approach to isolate CSC based on their ability to efflux Hoechst dye via ABC-transporters. This population is referred to as side population (SP). We consider the functional approach as less biased since it identifies the stem cells by their unique property of chemo-resistance. Given that epigenetic regulation plays a crucial role both in stem cell and cancer development, we chose epigenic modulation as an additional tool to narrow down the heterogeneity of SP-fraction. The specific objectives of this study are: (i) to characterize liver cancer stem cells heterogeneity using in vitro and in vivo functional assays;(ii) to investigate the effect of epigenetic modulation on the cancer stem cells by treatment with the DMNT1-inhibtior Zebularine (ZEB). Human HCC cell lines representing the different cell origins of primary liver cancer were selected for analysis including Huh7 (HCC), WRL68 (hepatoblastoma) and KMCH (mixed HCC/ICC). FACS analysis after Hoechst 33342-staining showed that the size of SP-fractions varied from 0.8% to 1.2%. In vitro treatment with 100 M ZEB for 3 days caused a significant reduction of the SP fraction in all three cell lines. In contrast, the colony-forming ability of SP cells as well as colony size was increased as measured by a quantitative soft-agar-based assay. ZEB treatment did not affect the viability of FACS sorted SP cells, although it reduced viability of the non-SP cells. To further assess tumorigenic potential of SP and non-SP cells, we transplanted 102-104 FACS sorted SP and non-SP cells into NOD/SCID mice. Consistent in all three HCC cell lines, an overall increase in tumor frequency was observed for SP cells regardless of ZEB treatment during the first 8 wk after transplantation. Limited dilution analysis (LDA) revealed a remarkable enrichment of tumor initiating cells within the SP fraction from all cell lines as compared to non-SP cells (3.1-fold, P=0.009 and 4.3-fold, P=0.001, at 6 and 8 wk, respectively). However at 10 wk, the differences became less pronounced although the tumor frequency in the group of mice receiving 100 SP cells remained higher (7/12 vs. 3/12 in non-SP). ZEB treatment significantly increased the number of tumor initiated cells in SP fraction. As few as 100 cells produced tumors in ZEB-treated SP cells from all cell lines (Figure 3, as shown for Huh7), while 1000 treated non-SP cells gave rise to few or no tumors (Huh7) within 20 weeks of observation. LDA analysis performed at 10 wk revealed a 15-fold increase in the tumor-initiating capacity of SP cells compared to non-SP (P=5.45x10x6). Treated SP cells showed a significant increase in tumorigenic potential as compared to their untreated counterpart (7-fold, P=0.001). Thus, ZEB-treatment significantly enhanced the tumorigenicity of the SP fraction while slightly reducing the tumor forming ability of non-SP fraction. To address the molecular mechanisms underlying these differences, we performed qRT-PCR analysis of selected genes. CSC-associated genes, such as ABCG2, CD133, GPC3 and c-KIT, as well as pluripotency related genes (OCT4, NANOG) were selectively overexpressed in the SP cells. Consistent with our in vivo and in vitro results, ZEB treatment significantly amplified the differences in the expression levels of CSC and stemness associated genes between SP and non-SP cells. These results suggest that (i) SP fraction is highly enriched for cells possessing tumor initiating ability and CSC properties;(ii) ZEB treatment can significantly reduce the heterogeneity of SP cells thus increasing the frequency of CSC;and (iii) the combination of an isolation procedure based on stem-like functional characteristics with epigenetic modulation provides an important tool for investigating cancer stem cells biology. Future plans include (i) to define antigenic characteristics of the cancer stem cells before and after ZEB treatment;(ii) to define specific gene expression signatures using microarray analysis;(iii) to integrate gene expression signatures with copy number variation (CNV) analysis obtained from the same groups;(iv) to characterize the effect of epigenetic modulation on the cancer stem cells by methylation specific PYRO-sequencing;(v) obtain primary human liver tumors to validate the ongoing work in cell lines;(vi) to test the usefulness of these integrative specific genomic signatures for prognostic prediction, both retrospectively and prospectively, in cohorts of HCC patients available to us at the National Cancer Institute (USA);and (vii) to employ the integrative genomic signatures for identification of novel and specific molecular therapeutic targets for the HCC derived CSC.